Unexpected transcriptome pompTʹ contributes to the increased pathogenicity of a pompT mutant of avian pathogenic Escherichia coli
Unexpected transcriptome pompTʹ contributes to the increased pathogenicity of a pompT mutant of avian pathogenic Escherichia coli
复制标题
意外的转录组 pompTÊ1 导致禽致病性大肠杆菌 pompT 突变体的致病性增加
作者：
Juanhua Liu;Xiaohui Mu;Xiaobo Wang;Haixia Huan;Qingqing Gao;Juan Chen;Peizhuang Qiao;Luyao Jiang;Song Gao;Xiufan Liu
λ red重组系统具有适用性广、简便、高效等特点，适合于禽致病性大肠杆菌（APEC）毒力基因的筛选。在APEC E058（O2血清群）中，存在两个拷贝的外膜蛋白酶（ompT）基因，compT编码位于染色体上的cOmpT，pompT编码位于ColV质粒上的pOmpT。然而，发病机制和pompT表达在APEC E058之间的关系尚未阐明。在这里，我们成功地构建了两个pompT基因突变体：E058 pompT含有氯霉素（猫）抗性基因和E058 pompT没有猫基因。通过RT-PCR和测序分析，在突变株E058 pompT中检测到一个意想不到的转录组pompT，该突变株在λ red重组系统诱导下缺失了cat基因。突变株E058ΔpompT在鸡感染模型和HD 11细胞模型中的致病性均较亲本株明显减弱，而突变株E058ΔpompT在鸡感染模型和HD 11细胞模型中的致病性无明显差异。此外，非预期转录组ompT β的存在影响SPF鸡血清的杀菌活性，并降低鸡心脏组织中TLR 2的转录水平。我们的研究确定pompT基因在APEC E058的毒力中起重要作用，并且意想不到的转录组ompT突变体有助于在λ red重组系统诱导的cat基因缺失后增加APEC E058突变体的致病性，这表明该系统在构建突变株特别是在活疫苗开发中使用的突变株时仍然存在一些局限性。
The lambda red recombination system makes it suitable for screening virulence gene utility in avian pathogenic Escherichia coli (APEC) on account of its wide applicability, simplicity and high efficiency. In APEC E058 (O2 serogroup) , there are two copies of the outer membrane protease (ompT) gene, compT encoding cOmpT that is located on the chromosome and pompT encoding pOmpT that is located on a ColV plasmid. However, the relationship between pathogenesis and pompT expression in APEC E058 has yet to be elucidated. Here, we successfully constructed two pompT gene mutants: E058pompT containing a chloramphenicol (cat) resistance gene and E058pompTʹ without the cat gene. By RT-PCR and sequencing analysis, an unexpected transcriptome pompTʹ was detected in mutant strain E058pompTʹ after deletion of the cat gene induced by the lambda red recombination system. Surprisingly, the pathogenicity of mutant E058ΔpompT was significantly attenuated compared to its parental strain in the chicken infection model and HD11 cell model then the pompT gene was knocked out, while the pathogenicity of the other mutant strain E058ΔpompTʹ had no difference. Furthermore, the presence of unexpected transcriptome ompTʹ influenced the bactericidal activity of SPF chicken serum and decreased the transcription level of TLR2 in the heart tissue of chickens. Our study identifies the pompT gene plays an important role in the virulence of APEC E058, and the unexpected transcriptome ompTʹ contributes to the increased pathogenicity of APEC E058 mutants following deletion of the cat gene induced by the lambda red recombination system, which suggests that this system still has some limitations for construction of mutant strains particularly where these are used in development of live vaccine.